The proliferation and differentiation of possible Leydig cell precursors in adult rats were studied after destruction of the existing Leydig cells with EDS or after daily treatment with hCG. After 2 days with either treatment, a 12- to 16-fold increase in the number of [3H]thymidine-incorporating interstitial cells was found. In the case of hCG treatment, this was probably due to the high plasma hCG levels. However, after EDS treatment, LH levels start to rise between days 1 and 3, suggesting a paracrine stimulation of the proliferation of interstitial cells. After hCG treatment, a substantial increase in the numbers of Leydig cells was already found at day 2. It was concluded that hCG induced a rapid differentiation, without cell division, of existing precursor cells into recognizable Leydig cells. In rats treated with both EDS and hCG, new Leydig cells were not formed during the first 10 days. This indicates that EDS destroys not only mature Leydig cells but also those Leydig cell precursors that are able to differentiate rapidly into recognizable Leydig cells.

Microplastics have been documented in drinking water, but their effects on human health from ingestion, or the concentrations at which those effects begin to manifest, are not established. Here, we report on the outcome of a virtual expert workshop conducted between October 2020 and October 2021 in which a comprehensive review of mammalian hazard studies was conducted. A key objective of this assessment was to evaluate the feasibility and confidence in deriving a human health-based threshold value to inform development of the State of California’s monitoring and management strategy for microplastics in drinking water. A tiered approach was adopted to evaluate the quality and reliability of studies identified from a review of the peer-reviewed scientific literature. A total of 41 in vitro and 31 in vivo studies using mammals were identified and subjected to a Tier 1 screening and prioritization exercise, which was based on an evaluation of how each of the studies addressed various quality criteria. Prioritized studies were identified largely based on their application and reporting of dose–response relationships. Given that methods for extrapolating between in vitro and in vivo systems are currently lacking, only oral exposure in vivo studies were identified as fit-for-purpose within the context of this workshop. Twelve mammalian toxicity studies were prioritized and subjected to a Tier 2 qualitative evaluation by external experts. Of the 12 studies, 7 report adverse effects on male and female reproductive systems, while 5 reported effects on various other physiological endpoints. It is notable that the majority of studies (83%) subjected to Tier 2 evaluation report results from exposure to a single polymer type (polystyrene spheres), representing a size range of 0.040 to 20 µm. No single study met all desired quality criteria, but collectively toxicological effects with respect to biomarkers of inflammation and oxidative stress represented a consistent trend. While it was possible to derive a conservative screening level to inform monitoring activities, it was not possible to extrapolate a human–health-based threshold value for microplastics, which is largely due to concerns regarding the relative quality and reliability of current data, but also due to the inability to extrapolate data from studies using monodisperse plastic particles, such as polystyrene spheres to an environmentally relevant exposure of microplastics. Nevertheless, a conservative screening level value was used to estimate a volume of drinking water (1000 L) that could be used to support monitoring activities and improve our overall understanding of exposure in California’s drinking water. In order to increase confidence in our ability to derive a human–health-based threshold value in the future, several research recommendations are provided, with an emphasis towards strengthening how toxicity studies should be conducted in the future and an improved understanding of human exposure to microplastics, insights critically important to better inform future risk assessments. Graphical abstract

Puberty in the male is dependent upon the elevated production of testosterone by the Leydig cells. LH affects this increase in testosterone output by increasing the total number of Leydig cells in the testis and by stimulating the steroidogenic pathway in these cells. Since Leydig cell proliferation is a prerequisite for the onset of puberty, we have examined the ability of LH and growth factors known to be present in the testis to promote DNA synthesis. Leydig cells were isolated from 21-day-old rats, cultured in serum-free medium for 48 h to become quiescent, and then treated with LH and growth factors for 18 h. [3H]Thymidine incorporation into DNA was assessed over the subsequent 4-h incubation period. Cells in control cultures incorporated low levels of [3H]thymidine into DNA and were stimulated after treatment with LH (100 ng/ml). Insulin/insulin-like growth factor-1 (IGF-1) and transforming growth factor-alpha (TGF-alpha), previously localized in Leydig cells by immunohistochemistry, also stimulated [2H]thymidine incorporation into DNA. The responses of the Leydig cells to maximum levels of insulin and TGF-alpha were dependent on the cell density. Insulin and TGF-alpha alone and in combination increased the number of cells labeled with [3H]thymidine, as assessed by autoradiography. TGF-beta, known to be secreted by Sertoli cells, also stimulated DNA synthesis under basal conditions, but the maximum response was significantly lower than that achieved in the presence of TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

There is a general agreement that granulosa cell apoptosis is the cause of antral follicle attrition. Less clear is whether this pathway is also activated in case of preantral follicle degeneration, as several reports mention that the incidence of granulosa cell apoptosis in preantral follicles is negligible. Our objective is therefore to determine which cell-death pathways are involved in preantral and antral follicular degeneration.Atretic preantal and antral follicles were investigated using immunohistochemistry and laser-capture microdissection followed by quantitative real-time reverse transcription polymerase chain reaction. Microtubule-associated light-chain protein 3 (LC3), sequestosome 1 (SQSTM1/P62), Beclin1, autophagy-related protein 7 (ATG7), and cleaved caspase 3 (cCASP3) were used as markers for autophagy and apoptosis, respectively. P62 immunostaining was far less intense in granulosa cells of atretic compared to healthy preantral follicles, while no difference in LC3 and BECLIN1 immunostaining intensity was observed. This difference in P62 immunostaining was not observed in atretic antral follicles. mRNA levels of LC3 and P62 were not different between healthy and atretic (pre)antral follicles. ATG7 immunostaining was observed in granulosa cells of preantral atretic follicles, not in granulosa cells of degenerating antral follicles. The number of cCASP3-positive cells was negligible in preantral atretic follicles, while numerous in atretic antral follicles. Taken together, we conclude that preantral and antral follicular atresia is the result of activation of different cell-death pathways as antral follicular degeneration is initiated by massive granulosa cell apoptosis, while preantral follicular atresia occurs mainly via enhanced granulosa cell autophagy.

Dogs of different ages without testicular diseases were evaluated to study possible age-related changes in hormone concentrations in serum. Dogs with testicular tumours were also investigated to study the relation between tumour type and hormone concentrations; in this study, dogs with Sertoli cell tumours, Leydig cell tumours and seminomas were included. We measured testosterone, oestradiol, LH, FSH and inhibin-like immunoreactivity concentrations in peripheral venous and testicular venous blood of these animals.In normal dogs there appeared to be no age-related changes in the concentrations of the investigated hormones, except for a significant age-related decrease in oestradiol concentrations in testicular venous blood (P<0·02). Dogs with a Sertoli cell tumour had greater oestradiol concentrations and inhibin-like immunoreactivity in both peripheral and testicular venous blood than did dogs without a neoplasm (P<0·05). Testosterone concentrations were reduced in dogs with Sertoli cell tumours, as were FSH and LH. Feminisation occurred in eight of 13 dogs with a Sertoli cell tumour and in two of 14 dogs with a Leydig cell tumour; it was accompanied by a significantly greater oestradiol concentration than in normal dogs and in dogs with Sertoli cell tumours without signs of feminisation. Dogs with a Leydig cell tumour had greater concentrations of oestradiol and inhibin-like immunoreactivity in both peripheral venous and testicular venous blood than did dogs without a neoplasm (P<0·05). The testosterone concentration in testicular venous blood of these dogs was lower than that in dogs with normal testes. The concentration of LH in peripheral venous blood was also reduced (P<0·05). Hormone concentrations in dogs with a seminoma were not different from those in normal dogs.It was concluded that seminomas are not endocrinologically active. In contrast, both Sertoli cell tumours and Leydig cell tumours can cause increased oestrogen production leading to signs of feminisation. These tumours also have considerable amounts of inhibin-like immunoreactivity, but only in Sertoli cell tumours does this result in a reduction in FSH concentrations, suggesting that Sertoli cell tumours secrete dimeric inhibin, whereas Leydig cell tumours presumably produce loose -subunits that cross-react in the inhibin assay but are not biologically active.

In hypophysectomized rats, 2 days after the administration of the cytotoxic drug ethane dimethyl sulphonate (EDS), the proliferative activity of Leydig cell precursors increased six‐fold. Thus, factors other than LH act locally to stimulate the proliferation of precursor cells after EDS. Twenty‐six days after EDS administration, neither cells with the morphological characteristics of Leydig cells nor histochemical enzyme activities, such as 3 beta‐HSD and alpha‐naphtyl esterase, could be detected in testis tissue. In hypophysectomized rats treated daily with hCG (100 iu) for 7 days, starting at 26 days after EDS, the number of Leydig cells was increased to 48 ± cells (per 1000 Sertoli cells), which is approximatley 4.5% of the intact control level. 3 beta‐HSD and alpha‐naphtyl esterase activity could be detected, and plasma testosterone levels had increased 15‐fold compared with the hypophysectomized controls. These results show that proliferation and some differentiation of precursor cells along the Leydig cell lineage can occur independent of LH, but the final stages of the differentiation process require hCG stimulation.

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3 or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered. In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic demands of modern hybrid sows have increased over the years, which increases the chance that sows enter a substantial negative energy balance (NEB) during lactation. This NEB can influence the development of follicles and oocytes that will give rise to the next litter. To study effects of a lactational NEB on follicular development, we used 36 primiparous sows of which 18 were subjected to feed restriction (3.25 kg/day) and 18 were full-fed (6.5 kg/day) during the last 2 weeks of a 24.1 ± 0.3 day lactation. Feed restriction resulted in a 70% larger lactational body weight loss and 76% higher longissimus dorsi depth loss, but similar amounts of backfat loss compared to the full fed sows. These changes were accompanied by lower plasma insulin-like growth factor 1 (IGF1) and higher plasma creatinine levels in the restricted sows from the last week of lactation onward. Ovaries were collected 48 h after weaning. Restricted sows had a lower average size of the 15 largest follicles (−26%) and cumulus–oocyte complexes showed less expansion after 22 h in vitro maturation (−26%). Less zygotes of restricted sows reached the metaphase stage 24 h after in vitro fertilization and showed a higher incidence of polyspermy (+89%). This shows that feed restriction had severe consequences on oocyte developmental competence. Follicular fluid of restricted sows had lower IGF1 (−56%) and steroid levels (e.g., β-estradiol, progestins, and androgens), which indicated that follicles of restricted sows were less competent to produce steroids and growth factors needed for oocytes to obtain full developmental competence.